Some very potent chemotherapeutics used in the clinic function by stabilizing covalent protein-DNA adducts formed as obligatory intermediates in normal cellular function. Despite the demonstrated effectiveness of these drugs, there has been no effort to systematically identify all targets of drugs in this class. Our proposal describes a novel approach that combines stringent enrichment of covalent protein adducts upon drug treatment with unbiased proteomics to identify and discover drug targets. Using this approach, we have confirmed known targets of topotecan and etoposide, and we have identified novel targets of these drugs, thereby establishing the power of this approach as a discovery tool. We propose to apply this approach to identify the targets of six chemotherapeutic drugs that function by this mechanism: etoposide, doxorubicin, topotecan, decitabine (5-aza-dC), azacitidine (5-aza-C) and olaparib, and to validate these targets. Success in these experiments will identify new targets of drugs in current use, and establish a general approach for determining mechanisms of drug action to enable future identification of novel drug-target pairs.